Tissue specificity of mitochondrial gene expression W D Richardsons lab Daniel Dilg BSc Biology The Mitochondrion Ancestor of a cyanobacteria that entered a eukaryotic cell a long time ago Powers the cell by turning ADP to ATP via a highly conserved electron transport chain Transfer of the symbionts genome to the nucleus gave rise to NMGs: Nuclear Mitochondrial Genes NMG families follow the law of eukaryotic gene regulation Why researching Nuclear Mitochondrial Genes? Mitochondrial encephalomyopathies (ATPAF2 / ATP12) Lebers hereditary optic neuropathy (ND4 & ND1: Mt DNA) Aminoglycoside-induced non-syndromic hearing loss (Mt DNA) Examples of Diseases: With only 16Kb, the mitochondrial genome seems easy to study. But there are thousands of mitochondria per cell, and each contain up to ten copies of its genome. Nuclear Mitochondrial Genes Dars1 (cytoplasmic) Dars2 (mitochondrial) Polg1 : Mt DNA polymerase catalytic subunit Polg2 : Mt DNA polymerase accessory subunit Atpaf2 : ATP synthase mitochondrial F1 complex assembly factor 2 tRNA Asp synthetases In situ Hybridization 1) Check the plasmid and digest it: 2) Run a transcription reaction with dNTPS and labelled Uracil: 3) Apply RNA probe on fixed sections of tissue Single stranded RNA RNA polymerase 4) Finally, detect localization of annealed RNA probed target RNA using antibodies and blue colour reaction The mouse embryo Sagittal section of an E15.5 embryo Lower abdomen transverse section 20m transverse sections Dars 2, E17.5, transverse section Dars 1, E17.5, Saggital section Because it is hard to compare and contrast different type of sections, the same probes were then tested on similar sections. Brown fat Cartilage of vertebra Origin of Sternothyroid Liver Gut Spinal chord Dars 2 Dorsal root Ganglia Liver Gut folding Dars 1 Spinal chord Results continued Lumen of midgut Liver Muscle fibers going to form right dome of diaphragm Deposit of Brown (multicolour) fat Lower lip Tip of tongue Entrance into nasopharinx E15.5, ATPAF2, Sagittal Discussion and future work Gut & Brown Fat Expression Liver & Dorsal Root Ganglia Specific Expression Dars2Dars1Genes The existence of false positive expression * Tissue can be sticky, probe stays on it even though the target RNA does not match * High cell density in muscle and fat, hence more signal DNA conformation (Secondary structure) prevents transcription or digestion no POLG1/2 probe Dars 1 and 2 expression In the future: Isolate a candidate tissue and perform Quantitative Reverse Transcription PCR to verify gene expression (Dars 2 Gut for example) Why is Dars 1 strongly expressed in the Dorsal root ganglia even though the cell density is very low? Avoid secondary structure for Polg probe by choosing other 3UTR region